Curcumin Inhibits Proliferation Of Oral Squamous Cell Carcinoma Through Regulating Prdx6 Expression

Objective:The incidence of oral squamous cell carcinoma（OSCC）is highest in the malignant tumor of head and neck.Surgery and chemotherapy are the major treatment because the pathogenesis of OSCC has not been fully elucidated.Its five-year survival rate is not more than 50%.Curcumin,as a natural compound extracted from curcuma longa,applied for the treatment of diseases including lung inflammation and cancer.Curcumin can inhibit the proliferation,angiogenesis,invasion and metastasis of tumor through various ways,and then interfere with the progression of tumor.However,the detailed mechanism of curcumin against tumor has not been entirely revealed.Peroxiredoxin 6（Prdx6）,as one of important members in peroxidase family,has both peroxidase and phospholipase A₂（PLA₂）activities.Prdx6 is highly expressed in tissue of OSCC,indicating the potential important role of Prdx6 in the progression of OSCC.This study is to investigate the regulation of Prdx6 by curcumin and its role in the proliferation of OSCC,which may provide new evidence and data for further elucidation of anti-tumor mechanism of curcumin,and prevention and treatment of OSCC.Methods:CAL27 cells were used in this study.Curcumin with different concentrations（0,10μmol/L,20μmol/L,40μmol/L,80μmol/L,160μmol/L）,Prdx6 siRNA and MJ33,an inhibitor of PLA₂ activity,were used to intervene.MTS assay was used to detect the cell viability of CAL27 cells.Cell clone formation assay was used to detect the proliferation of CAL27 cells.Flow cytometry was used to detect the apoptotic rate of CAL27 cells.DCFH-DA probe was used to detect the level of reactive oxygen species in CAL27 cells.Quantitative Real-time PCR was used to detect the expression of Prdx6 gene.Western blot was used to detect the expression of Prdx6 protein.Results:1.The cell viability of CAL27 cells was significantly reduced in a dose-dependent at 24h,48h and 72h under the treatment of curcumin with different concentrations（0,10μmol/L,20μmol/L,40μmol/L,80μmol/L,160μmol/L）.The clone formation number and size of CAL27 cells was significantly reduced at 10d under the treatment of 40μmol/L curcumin.The apoptotic rate of CAL27 cells was significantly increased at 48h under the treatment of 40μmol/L curcumin.The ROS level of CAL27 cells was increased in a dose-dependent at 6h under the treatment of curcumin with different concentrations（0,20μmol/L,40μmol/L,80μmol/L）2.The cell viability of CAL27 cells was significantly reduced at 24h under the treatment of 40μmol/L MJ33.The clone formation number and size of CAL27 cells was significantly reduced in a dose-dependent at 10d under the treatment of MJ33with different concentrations（0,10μmol/L,20μmol/L,40μmol/L）.The apoptotic rate and ROS level of CAL27 cells were significantly increased at 6h and 24h under the treatment of MJ33 with different concentrations（0,40μmol/L,60μmol/L,80μmol/L）.3.The results of fluorescence microscopy showed that the transfection efficiency of Prdx6 siRNA was about 80%.The results of Quantitative Real-time PCR showed that the Prdx6 gene expression level of CAL27 cells was reduced by about 70%compared with the negative control group at 36h under the transfection of Prdx6siRNA.The results of Western blot showed that the Prdx6 protein expression level of CAL27 cells was reduced by about 50%compared with the negative control group at48h under the transfection of Prdx6 siRNA.4.The cell viability of CAL27 cells was significantly reduced compared with the negative control group at 48h under the transfection of Prdx6 siRNA.The ROS level of CAL27 cells was significantly increased compared with the negative control group at 48h under the transfection of Prdx6 siRNA.The clone formation number and size of CAL27 cells was significantly reduced compared with the negative control group at10d under the transfection of Prdx6 siRNA.5.The results of Quantitative Real-time PCR showed that the Prdx6 gene expression level of CAL27 cells was significantly decreased at 6h under the treatment of 40μmol/L curcumin.The result of Western blot showed that the Prdx6 protein expression level of CAL27 cells was significantly decreased at 24h under the treatment of 40μmol/L curcumin.Conclusion:Curcumin increases the level of ROS and apoptosis of tumor cells and inhibits proliferation of OSCC by down-regulating Prdx6 expression.